The adult mammalian heart shows little or no significant natural regeneration of cardiac muscle after injury, and instead heals by scarring. This regenerative deficiency has enormous socioeconomic consequences, given that ischemic myocardial infarction is the leading cause of morbidity and mortality in the United States and over 5 million Americans suffer from heart failure. A decade ago, we found that the teleost zebrafish displays a robust regenerative response after partial resection of the cardiac ventricle, involving creation of new cardiomyocytes with little or no scarring. Pre-existing cardiomyocytes, not stem cells, are the primary source of new muscle during heart regeneration in zebrafish. Muscle regeneration is influenced by activities of the epicardium and endocardium, major non-muscle cells that line the cardiac chambers. There remain critical roadblocks to understanding how and why cardiac regeneration occurs. First, we know little about the regulatory mechanisms that control the competency of adult cardiomyocytes to proliferate in response to injury. Second, our understanding of the cellular and molecular components that stimulate regeneration by cardiomyocytes is insufficient. The goals of the current application are to address central questions about the source and stimuli for bona fide heart regeneration, using state-of-the-art screening and analysis tools in the zebrafish model system. 1) We will define chromatin regulatory signatures of heart regeneration by genome-wide profiling, and harness this information to identify enhancer elements that control gene expression during regeneration. 2) We will define the regenerative capacity of the epicardium, using new tools for visualizing and manipulating this important structure. 3) We will identify small molecule modulators of heart regeneration, using a new in vivo screening strategy that visualizes proliferating cardiomyocytes in live zebrafish. With these approaches, we will test the hypothesis that specialized injury cues activate dedicated cardiac muscle regeneration programs. Our work will provide a detailed understanding of cardiac regeneration and identify key regulators. These discoveries will inform approaches for comprehending and enhancing the limited regenerative response displayed by humans after myocardial infarction.